U.S. patent number 6,207,702 [Application Number 09/441,644] was granted by the patent office on 2001-03-27 for method for reducing postprandial oxidative stress using cocoa procyanidins.
This patent grant is currently assigned to Mars, Incorporated. Invention is credited to Leo J. Romanczyk, Jr., Harold H. Schmitz.
United States Patent |
6,207,702 |
Schmitz , et al. |
March 27, 2001 |
Method for reducing postprandial oxidative stress using cocoa
procyanidins
Abstract
A method for reducing postprandial oxidative stress and
associated pathologies by the dietary intake of cocoa procyanidins,
such as epicatechin is disclosed.
Inventors: |
Schmitz; Harold H. (Branchburg,
NJ), Romanczyk, Jr.; Leo J. (Hackettstown, NJ) |
Assignee: |
Mars, Incorporated (McClean,
VA)
|
Family
ID: |
23753716 |
Appl.
No.: |
09/441,644 |
Filed: |
November 17, 1999 |
Current U.S.
Class: |
514/453; 424/776;
426/479; 426/593; 426/631; 426/655; 426/72; 426/804; 514/456 |
Current CPC
Class: |
A23G
1/30 (20130101); A23G 1/426 (20130101); A61K
31/353 (20130101); Y10S 426/804 (20130101) |
Current International
Class: |
A23G
1/00 (20060101); A61K 31/352 (20060101); A61K
31/353 (20060101); A61K 031/35 () |
Field of
Search: |
;424/195.1 ;514/453,456
;426/72,593,479,631,655,804 |
References Cited
[Referenced By]
U.S. Patent Documents
Primary Examiner: Krass; Frederick
Attorney, Agent or Firm: Kelley; Margaret B. Clifford Chance
Rogers & Wells, LLP
Claims
What is claimed is:
1. A method for treating a mammal suffering from or at risk to
suffer from atherosclerosis, by reducing postprandial oxidative
stress in the mammal, which method comprises having the mammal
consume an effective amount of a cocoa polyphenol.
2. The method of claim 1, wherein the cocoa polyphenol is cocoa
procyanidins.
3. The method of claim 2, wherein the cocoa procyanidins are
monomers and/or oligomers of catechin and epicatechin.
4. The method of claim 3, wherein the oligomers are dimers through
octadecamers.
5. The method of claim 1, wherein the cocoa polyphenols are present
in a food, a dietary supplement, or a pharmaceutical.
6. The method of claim 5, wherein the food is a beverage.
7. The method of claim 5, wherein the food is a confectionery.
8. The method of claim 5, wherein the dietary supplement further
comprises a nutrient or a carrier.
9. The method of claim 5, wherein the pharmaceutical further
comprises a carrier, a diluent, or an excipient.
10. The method of claim 7, wherein the confectionery is a
chocolate.
11. The method of claim 5, wherein the food is a chocolate prepared
using cocoa ingredients which are prepared from cocoa beans or
blends thereof having a fermentation factor of 275 or less.
12. The method of claim 11, wherein the cocoa ingredients are
selected from the group consisting of chocolate liquor, cocoa
solids, roasted cocoa nibs or nib fractions, or a solvent-derived
cocoa extract.
13. The method of claim 10, wherein the chocolate is a dark
chocolate.
14. The method of claim 13, wherein the dark chocolate is a
bittersweet, semisweet, or sweet dark chocolate.
15. The method of claim 14, wherein the chocolate is a Standard of
Identity chocolate.
16. The method of claim 14, wherein the chocolate is a non-Standard
of Identity chocolate.
17. The method of claim 10, wherein the chocolate is a milk
chocolate, buttermilk chocolate, skim milk chocolate or mixed dairy
milk chocolate.
18. The method of claim 10, wherein the chocolate is a reduced-fat
chocolate.
19. The method of claim 17, wherein the chocolate is a Standard of
Identity chocolate.
20. The method of claim 17, wherein the chocolate is a non-Standard
of Identity chocolate.
21. The method of claim 10, wherein the chocolate is a white
chocolate or a white chocolate coating.
22. The method of claim 11, wherein the cocoa ingredients are
prepared from underfermented beans or mixtures of fermented beans
and underfermented and/or unfermented beans.
23. A method of reducing the risk of pathologies associated with
oxidative stress in a mammal suffering from atherosclerosis, which
method comprises having the mammal consume an amount of a cocoa
polyphenol effective to reduce oxidative stress.
24. The method of claim 23, wherein the associated pathologies are
selected from the group consisting of coronary heart diseases,
neurodegenerative disorders, and cancer.
25. The method of claim 2, wherein the cocoa procyanidins comprise
monomeric and/or oligomeric fractions.
26. The method of claim 25, wherein the cocoa procyanidins are
pooled fractions.
27. The method of claim 5, wherein the food, the dietary
supplement, or the pharmaceutical is prepared using cocoa
ingredients prepared from slaty cocoa beans, purple cocoa beans,
mixtures of slaty and purple cocoa beans, mixtures of purple and
brown cocoa beans, or mixtures of slaty, purple, and brown cocoa
beans.
28. The method of claim 5, wherein during the preparation of the
food, milk and/or sweetener is protected by pretreatment with an
antioxidant, an emulsifier, a fat, a flavorant, or mixtures
thereof.
29. The method of claim 28, wherein the pretreated ingredients
comprise a mixture of cocoa butter and lecithin.
30. The method of claim 29, wherein the food is a pet food, a dry
cocoa mix, a pudding, a syrup, a cookie, a savory sauce, a rice
mix, a rice cake, a cocoa beverage, a carbonated beverage, a
chocolate confectionery, a cocoa and a nut based product, a
nut-based product, or a low-fat food.
31. The method of claim 30, wherein the chocolate confectionery is
a reduced fact chocolate, a dark chocolate, a milk chocolate, or a
white chocolate.
32. A method of treating a mammal suffering from or at risk of
suffering from post-prandial oxidative stress by administering to
the mammal an amount of at least one cocoa polyphenol effective to
reduce post-prandial oxidative stress.
Description
FIELD OF THE INVENTION
This invention relates to a method for reducing postprandial
oxidative stress.
BACKGROUND OF THE INVENTION
Studies have linked certain dietary factors with atherosclerosis, a
forerunner of coronary heart disease (Addis, P. B., Carr, T. P.,
Hassel, C. A., Hwang, Z. Z., Warner, G. J., Atherogenic and
anti-atherogenic factors in the human diet. Biochem. Soc. Symp. 61,
259-271 (1995)). For example, a diet high in polyunsaturated fatty
acids (PUFAS) may render low-density lipoprotein (LDL) more
susceptible to peroxidation (Addis et al. 1995). The peroxidation
of LDL can cause tissue damage leading to atherosclerosis
(Sarkkinen, E. S., Uusitupa, M. I. J., Nyyssonen, K., Parviainen,
M., Penttila, I., Salonen, J. T., Effects of two low-fat diets,
high and low in polyunsaturated fatty acids, on plasma lipid
peroxides and serum vitamin E levels in free-living
hypercholesterolaemic men. European Journal of Clinical Nutrition
(1993) 47: 623-630). The peroxidation of LDL is a result of the
neutrophilic production of a superoxide anion radical or other
reactive species (Steinberg, D., Parthasapathy, S., Carew, T. E.,
Khoo, J. C., Witztum, J. L. (1989) Beyond cholesterol.
Modifications of low-density lipoprotein that increases its
atherogenicity. New England Journal of Medicine 320: 915-924). The
reactive species produced interact with PUFAS to form lipid peroxyl
radicals, which subsequently produce lipid hydroperoxides and
additional lipid peroxyl radicals (Steinberg et al. 1989). This
initiates a peroxidative cascade which may eventually modify an
essential part of the lipid's membrane, causing changes in membrane
permeability and even cell death (Steinberg et al. 1989).
Peroxidative degradation of LDL also leads to the formation of
lipid oxidation products such as malondialdehyde (MDA) and other
aldehydes which may be potentially toxic to the cell (Steinberg et
al. 1989).
Oxidative stress has been implicated in a variety of diseases and
pathological conditions, including endothelial cell cytotoxicity,
coronary heart diseases (such as thrombosis and hyperlipemia) and
cancer. (Addis et al. 1995). Recent studies have shown that
elevated lipid peroxidation levels (oxidative stress) may play a
role in the pathogenesis of Alzheimer's disease which includes a
group of neurodegenerative disorders with diverse etiologies, but
the same hallmark brain lesions. Practico D. et al., Increased
F2-isoprostanes in Alzheimer's disease: evidence for enhanced lipid
peroxidation in vivo. FASEB J. 1998 Dec.; 12 (15): 1777-1783.
Clinical studies have established that elevated plasma
concentrations of LDL are associated with atherosclerosis, a most
prevalent cardiovascular disease and the principle cause of heart
attack, stroke and vascular circulation problems (Sarkkinen et al.
1993). It is believed that a reduction of atherogenic lipid
peroxides (which are transported in the LDL fraction of blood
serum) can reduce the risk of atherogenesis (Mazur, A., Bayle, D.,
Lab, C., Rock, E., Rayssiguier, Y., Inhibiting effect of
procyanidin-rich extracts on LDL oxidation in vitro.
Atherosclerosis 145 (1999) 421-422). Antioxidants limit oxidative
modification of LDL and consequently lower plasma concentrations of
LDL, thereby acting as anti-atherogenic compounds (Sarkkinen et al.
1993). The oxidation of LDL has been reported as a model for
testing the ability of polyphenols to act as antioxidants by
breaking the peroxidative cascade described above (Rice-Evans, C.,
Plant polyphenols: free radical scavengers or chain-breaking
antioxidants? Biochem. Soc. Symp. 61, 103-116 (1995)). Studies have
reported that polyphenols can break the chain of the peroxidative
process by intercepting free radicals before they reenter the cycle
(Rice-Evans 1995).
SUMMARY OF THE INVENTION
This invention is directed to a method for reducing postprandial
oxidative stress and associated pathologies by the dietary intake
of cocoa polyphenols, including cocoa procyanidins. Cocoa
procyanidins include monomers and dimers of catechin and
epicatechin.
Cocoa procyanidins can be obtained from several Theobroma cacao
genotypes by the procedures discussed hereinafter. Cocoa
procyanidins can also be obtained by synthetic methods described in
PCT/US98/21392 (published as WO 99/19319 on Apr. 22, 1999) which is
incorporated herein by reference. The oligomers synthesized using
these methods may be linear, having the structure: ##STR1##
where X is an integer from zero to sixteen or branched, having the
structure: ##STR2##
where A and B are independently integers from one to fifteen.
It has been found that the dietary intake of an effective amount of
cocoa procyanidins counteracts postprandial oxidative stress which
has been linked to associated pathologies as described herein.
Postprandial oxidative stress occurs following the ingestion of
food products and has been linked with hyperlipidemia and increased
risk of cardiovascular disease. Ursini F. et al., Postprandial
plasma lipid hydroperoxides: a possible link between diet and
atherosclerosis. Free Radic. Biol. Med. 1998 Jul. 15; 25 (2):
250-252. Consequently, the dietary intake of an effective amount of
cocoa procyanidins counteracts these pathologies associated with
postprandial oxidative stress.
Measuring the formation of lipid oxidative products is one way to
assay oxidative stress. Cocoa procyanidins reduce LDL peroxidation
which consequently reduces the formation of lipid oxidation
products which can be assayed as described herein. One such lipid
oxidation product is malondialdehyde (MDA) which may be potentially
toxic to the cell. Cocoa procyanidins can be found in foods common
in the human diet, including chocolate. Epicatechin is a cocoa
procyanidin abundant in chocolate.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the nanomoles (nmol) of malondialdehyde (MDA) in
plasma at 2 and at 6 hours following ingestion of 1/2 bagel and a
dark chocolate product which was made with enhanced levels of cocoa
polyphenols and following ingestion of 1/2 bagel and a control
chocolate containing lower levels cocoa polyphenols, including
cocoa procyanidins (CPs).
FIG. 2 shows the nanomoles (nmol) of malondialdehyde (MDA) in
plasma at 2 and at 6 hours following ingestion of 1/2 bagel alone
and following ingestion of 1/2 bagel with increasing quantities of
semisweet chocolate which is typically high in cocoa polyphenols,
including cocoa procyanidins (CPs).
DETAILED DESCRIPTION OF THE INVENTION
It has been found that the dietary intake of cocoa procyanidins
counteracts oxidative stress as measured by reduction of LDL
peroxidation. Consequently, there was reduction in the formation of
LDL peroxidation products, such as malondialdehyde (MDA), which may
be potentially toxic to the cell. Plasma lipid peroxides were
measured photometrically using a thiobarbituric acid (TBA) reaction
based on methods described in Yagi, K., Assay for blood plasma or
serum, Methods in Enzymology 105: 328-331 (1984) Academic Press,
Inc., Orlando, Fla. (Ed. L. Packer). MDA is a low molecular weight
end-product that forms via decomposition of the products formed by
lipid peroxidation. The MDA found in the plasma can be quantified
using the Yagi et al. methods because at low pH and elevated
temperature, MDA reacts with TBA to generate a fluorescent red
adduct of MDA and TBA (1:2 ratio). The fluorescent intensity of the
MDA:TBA adduct, which can be accurately quantified, parallels the
concentration of the adduct. Hence, the amount of lipid peroxide
produced can be fluorometrically measured using the TBA reaction,
using an MDA standard. Substances other than the lipid peroxides
can react with TBA and thereby distort results. These water-soluble
substances are eliminated from the plasma sample by isolating the
lipids using precipitation along with the serum protein using a
phosphotungstic acid-sulfuric acid system.
As shown below, levels of MDA decreased at 2 and at 6 hours
following ingestion of semisweet chocolate high in cocoa
polyphenols. Similarly, MDA levels decreased at 2 and at 6 hours
following ingestion of dark chocolate high in cocoa polyphenols.
The decreases were more pronounced when the intake of chocolate was
increased. MDA levels also decreased (albeit not as much) when
tested at 6 hours following ingestion of a dark chocolate which
contained less of the cocoa polyphenols (that is, lower amounts of
cocoa polyphenols than contained in the test chocolates). All of
the chocolates used in the experiments described herein were made
using the methods discussed hereinafter. All test products
contained enriched levels of cocoa procyanidins.
For example, the dark chocolate test product contained 147 mg total
cocoa procyanidins (40.6 mg monomer) per 36.9 gram test product.
The dark chocolate control product contained only 3.3. mg cocoa
procyanidins (1.8 mg monomers) per 36.9 gram control product. The
semisweet products contained 185 mg total cocoa procyanidins (45.3
mg monomers) per a 35 gram bag of semisweet chocolate bits. A
single bag serving was consumed as the single dosage size. A two
bag serving (70 grams) of semisweet chocolate bits product
contained 370 mg total cocoa procyanidins and a three bag serving
(105 grams) of semisweet chocolate bits product contained 555 mg
total cocoa procyanidins.
The quantities of cocoa procyanidin monomers and oligomers in the
test products were measured by the analytical methods discussed
hereinafter. Procyanidin levels were determined by analyzing levels
of chocolate liquor or jet black cocoa powder and calculating the
percentage of powder in the final product. The low levels of
procyanidins in the control dark chocolate product precluded direct
analysis.
The chocolate liquor used to make the test products and the control
product was a blend of cocoa beans, some of which were
underfermented. The beans were prepared by the methods described in
PCT/US97/15893 (published as WO 98/09533 on Mar. 12, 1998), which
is herein incorporated by reference. Standard of Identity rules
governed the different levels of chocolate liquor and sugar which
were used to prepare semisweet versus dark chocolate. The semisweet
chocolate had higher levels of chocolate liquor and sugar. The
semisweet chocolate and the dark chocolate test products were used
to demonstrate that even though the cocoa procyanidins were
delivered using two different forms of test products, similar
effects were exhibited by each.
Methods for preparing cocoa mass are described in U.S. Pat. No.
5,554,645 (issued Sep. 10, 1996) which is herein incorporated by
reference. Harvested cocoa pods were opened and the beans with pulp
were removed for freeze-drying. The pulp was manually removed from
the freeze-dried mass and the beans were subjected to the following
manipulations. The freeze-dried cocoa beans were first manually
dehulled and ground to a fine powdery mass with a TEKMAR Mill. The
resultant mass was then defatted overnight by Soxhlet extraction
using redistilled hexane as the solvent. Residual solvent was
removed from the defatted mass by vacuum at ambient
temperature.
The chocolate liquor and/or cocoa solids can be prepared by
roasting the cocoa beans to an internal bean temperature of
95.degree. C. to 160.degree. C., winnowing the cocoa nibs from the
roasted cocoa beans, milling the roasted cocoa nibs into the
chocolate liquor and optionally recovering cocoa butter and
partially defatted cocoa solids from the chocolate liquor. The
cocoa solids can be further defatted using conventional
methods.
Alternatively, partially defatted cocoa beans having a high cocoa
polyphenol content, i.e., a high cocoa procyanidin content, can be
obtained by processing without a bean or nib roasting step and
without milling the beans to chocolate liquor. Even higher levels
can be achieved if underfermented cocoa beans are used in this
process. This method conserves the cocoa polyphenols because it
omits the traditional roasting step. The method consists
essentially of the steps of: (a) heating the cocoa beans to an
internal bean temperature just sufficient to reduce the moisture
content to about 3% by weight and loosen the cocoa shell, typically
using a infra red heating apparatus for about 3 to 4 minutes; (b)
winnowing the cocoa nibs from the cocoa shells; (c) screw pressing
the cocoa nibs; and (d) recovering the cocoa butter and partially
defatted cocoa solids which contain cocoa polyphenols including
cocoa procyanidins. Optionally, the cocoa beans are cleaned prior
to the heating step, e.g., in an air fluidized bed density
separator. Preferably, the cocoa beans are heated to an internal
bean temperature of about 100.degree. C. to about 110.degree. C.,
more preferably less than about 105.degree. C. The winnowing can be
carried out in an air fluidized bed density separator. The above
process of heating the cocoa beans to reduce the moisture content
and loosen the cocoa shell is disclosed in U.S. Pat. No. 6,015,913
issued Jan. 18, 2000 (to K. S. Kealey, et al.). which is herein
incorporated by reference.
The internal bean temperature (IBT) can be measured by filling an
insulated container such as a thermos bottle with beans
(approximately 80-100 beans). In order to maintain the temperature
of the beans during transfer from the heating apparatus to the
thermos, the insulated container is then appropriately sealed in
order to maintain the temperature of the sample therein. A
thermometer is inserted into the bean filled insulated container
and the temperature of the thermometer is equilibrated with respect
to the beans in the thermos. The temperature reading is the IBT
temperature of the beans. IBT can also be considered the
equilibrium mass temperature of the beans.
The cocoa beans can be divided into four categories based on their
color: predominately brown (fully fermented), purple/brown, purple,
and slaty (unfermented). Preferably, the cocoa solids are prepared
from underfermented cocoa beans, i.e., slaty cocoa beans, purple
cocoa beans, mixtures of slaty and purple cocoa beans, mixtures of
purple and brown cocoa beans, or mixtures of slaty, purple, and
brown cocoa beans. More preferably, the cocoa beans are slaty
and/or purple cocoa beans have a higher cocoa polyphenol content
than fermented beans.
The cocoa polyphenol content of cocoa ingredients, for example, the
roasted cocoa nibs, chocolate liquor and partially defatted or
nonfat cocoa solids, is higher when the cocoa beans or blends
thereof have a fermentation factor of 275 or less. Preferably,
these cocoa beans are used for processing into cocoa ingredients.
The "fermentation factor" is determined using a grading system for
characterizing the fermentation of the cocoa beans. For example,
slaty beans are designated 1, purple beans as 2, purple/brown beans
as 3, and brown beans as 4. The percentage of beans falling within
each category is multiplied by the weighted number. Thus, the
"fermentation factor" for a sample of 100% brown beans would be
100.times.4 or 400, whereas for a 100% sample of purple beans it
would be 100.times.2 or 200. A sample of 50% slaty beans and 50%
purple beans would have a fermentation factor of 150
[(50.times.1)+(50.times.20)].
Conventional processing techniques do not provide food products,
especially confectioneries which adequately retain the cocoa
polyphenol concentrations. However, high cocoa polyphenol food
products may be prepared using conventional chocolate liquors or
these high cocoa polyphenol chocolate liquors and/or conventional
chocolate cocoa solids or high cocoa polyphenol cocoa solids by
protecting the milk and/or sweetener with a pretreatment ingredient
selected from the group consisting of an antioxidant, an
emulsifier, a fat, a flavorant and mixtures thereof, before adding
the cocoa ingredient. Preferred pretreatment ingredient is a
mixture of cocoa butter and lecithin.
Examples of high cocoa polyphenol food products include pet food,
dry cocoa mixes, puddings, syrups, cookies, savory sauces, rice
mixes and/or rice cakes, beverages, including cocoa beverages and
carbonated beverages. Preferably, the high cocoa polyphenol foods
are chocolate confectioneries, for example, dark chocolate,
semisweet chocolate, sweet chocolate, milk chocolate, buttermilk
chocolate, skim milk chocolate, mixed dairy milk chocolate and
reduced fat chocolate. Cocoa polyphenols may be added to white
chocolate and white chocolate coating to create products with high
levels of cocoa polyphenols. These confectioneries may be either
Standard of Identity chocolates or non-Standard of Identity
chocolates. Preferable non-chocolate food products include
nut-based products such as peanut butter, peanut brittle and the
like. Also included are low-fat food products prepared with
defatted or partially defatted nut meats. Cocoa procyanidins are
also used in dietary supplements and pharmaceuticals. Also included
are food products comprising at least one cocoa polyphenol and
L-arginine. The procyanidin and L-arginine may be provided,
respectively, by cocoa and/or nut procyanidins and an L-arginine
containing component, such as a nut meat. The L-arginine may be
derived from any available arginine source, e.g., Arachis hypogaea
(peanuts), Juglans regia (walnuts), Prunus amygdalus (almonds),
Corylus avellana (hazelnuts), Glycine max (soy bean) and the like.
The nut may be nut pieces, a nut skin, a nut paste, and/or a nut
flour present in amounts which provide the desired amount of
L-arginine, which will vary depending upon the nut source. The
L-arginine-containing ingredient may also be a seed, a seed paste,
and/or a seed flour. The cocoa polyphenols, including cocoa
procyanidins, may be synthetic or natural. The procyanidins may
from a source other than cocoa beans.
The food product may contain polyphenols, such as procyanidins,
from a source other than cocoa, e.g., the polyphenols found in the
skins of nuts such as those described above. Peanut skins contain
about 17% procyanidins, and almond skins contain up to 30%
procyanidins. In a preferred embodiment, the nut skins are used in
the food product, e.g., the nougat of a chocolate candy.
Polyphenols from fruits and vegetables may also be suitable for use
herein. It is known that the skins of fruits such as apples and
oranges, as well as grape seeds, are high in polyphenols.
As used herein "food" is a material consisting of protein,
carbohydrate and/or fat, which is used in the body of an organism
to sustain growth, repair vital processes, and to furnish energy.
Foods may also contain supplementary substances, such as, minerals,
vitamins, and condiments (Merriam-Webster Collegiate Dictionary,
10.sup.th Edition, 1993).
As used herein "food supplement" is a product (other than tobacco)
that is intended to supplement the diet that bears or contains one
or more of the following dietary ingredients: a vitamin, a mineral,
an herb or other botanical, an amino acid, a dietary substance for
use by man to supplement the diet by increasing the total daily
intake, or a concentrate, metabolite, constituent, extract or
combination of these ingredients. (Merriam-Webster Collegiate
Dictionary, 10.sup.th Edition, 1993). When the term is used on food
labels, "supplement" means that nutrients have been added in
amounts greater than 50% above the U.S. Recommended Daily Allowance
("Understanding Normal and Clinical Nutrition, 3.sup.rd Edition,
Editors Whitney, Cataldo and Rolfes at page 525).
As used herein "pharmaceutical" is a medicinal drug.
(Merriam-Webster Collegiate Dictionary, 10.sup.th Edition,
1993).
The cocoa procyanidins in these products are part of a larger
family of cocoa polyphenols which are present in cocoa beans.
Suitable cocoa procyanidin-containing ingredients include roasted
cocoa nibs or fractions thereof, chocolate liquor, partially
defatted cocoa solids, nonfat cocoa solids, cocoa powder milled
from the cocoa solids, and mixtures thereof. Preferably, the
ingredients are prepared from underfermented beans since these
beans contain higher amounts of cocoa polyphenols including the
cocoa procyanidins. Cocoa procyanidins can be obtained from several
Theobroma cacao genotypes which represent the three recognized
horticultural races of cocoa, namely, Trinitario, Forastero and
Criollo. See Engels, J. M. M., Genetic Resources of Cacao: A
catalogue of the CATIE collection, Tech. Bull. 7, Turrialba, Costa
Rica (1981). An extract containing cocoa polyphenols, including
cocoa procyanidins, can be prepared by solvent extracting the
partially defatted cocoa solids prepared from the underfermented
cocoa beans or cocoa nibs having a fermentation factor of 275 or
less, as described herein.
METHODS
Analytical Methods for the Quantification of Cocoa Procyanidins
The analytical method described below was used to separate and
quantify, by degree of polymerization, the procyanidin composition
of the seeds from Theobroma cacao and of chocolate. The analytical
method described below is based upon work reported in Hammerstone,
J. F., Lazarus, S. A., Mitchell, A. E., Rucker R., Schmitz H. H.,
Identification of Procyanidins in Cocoa (Theobroma cacao) and
Chocolate Using High-Performance Liquid Chromatography/Mass
Spectrometry, J. Ag. Food Chem.; 1999; 47 (10) 490-496. The utility
of the analytical method described below was applied in a
qualitative study of a broad range of food and beverage samples
reported to contain various types of proanthocyanidins, as reported
in Lazarus, S. A., Adamson, G. E., Hammerstone, J. F., Schmitz, H.
H., High-performance Liquid Chromatography/Mass Spectrometry
Analysis of Proanthocyanidins in Foods and Beverages, J. Ag. Food
Chem.; 1999; 47 (9); 3693-3701. The analysis in Lazarus et al.
(1999) reported analysis using fluorescence detection because of
higher selectivity and sensitivity.
Composite standard stock solutions and calibration curves were
generated for each procyanidin oligomer through decamer using the
analytical method described below, as reported in Adamson, G. E.,
Lazarus, S. A., Mitchell, A. E., Prior R. L., Cao, G., Jacobs, P.
H., Kremers B. G., Hammerstone, J. F., Rucker R., Ritter K. A.,
Schmitz H. H., HPLC Method for the Quantification of Procyanidins
in Cocoa and Chocolate Samples and Correlation to Total Antioxidant
Capacity, J. Ag. Food Chem.; 1999; 47 (10) 4184-4188. Samples were
then compared with the composite standard to accurately determine
the levels of procyanidins.
Extraction
The fresh seeds (from Brazilian cocoa beans) were ground in a
high-speed laboratory mill with liquid nitrogen until the particle
size was reduced to approximately 90 microns. Lipids were removed
from 220 grams (g) of the ground seeds by extracting three times
with 1000 milliliters (mL) of hexane. The lipid free solids were
air dried to yield approximately 100 g of fat-free material. A
fraction containing procyanidins was obtained by extracting with
1000 mL of 70% by volume acetone in water. The suspension was
centrifuged for 10 minutes at 1500 g. The acetone layer was
decanted through a funnel with glass wool. The aqueous acetone was
then re-extracted with hexane (.about.75 mL) to remove residual
lipids. The hexane layer was discarded and the aqueous acetone was
rotary evaporated under partial vacuum at 40.degree. C. to a final
volume of 200 mL. The aqueous extract was freeze dried to yield
approximately 19 g of acetone extract material.
Gel Chromatograhy
Approximately 2 g of acetone extract (obtained above) was suspended
in 10 mL of 70% aqueous methanol and centrifuged at 1500 g. The
supernatant was semi-purified on a Sephadex LH-20 column
(70.times.3 centimeters) which had previously been equilibrated
with methanol at a flow rate of 3.5 mL/min. Two and a half hours
after sample loading, fractions were collected every 20 minutes and
analyzed by HPLC for theobromine and caffeine See Clapperton, J.,
Hammerstone, J. F., Romanczyk, L. J., Yow, S., Lim, D., Lockwood,
R., Polyphenols and Cocoa Flavour, Proceedings, 16.sup.th
International Conference of Groupe Polyphenols, Lisbon, Portugal,
Groupe Polyphenols: Norbonne, France, 1992; Tome II, pp. 112-115.
Once the theobromine and caffeine were eluted off the column
(.about.3.5 hours), the remaining eluate was collected for an
additional 4.5 hours and rotary evaporated under partial vacuum at
40.degree. C. to remove the organic solvent. Then the extract was
suspended in water and freeze dried.
Purification of Procyanidin Oligomers by Preparative Normal-Phase
HPLC
The cocoa extract from above (0.7 g) was dissolved in (7 mL)
mixture of acetone/water/acetic acid in a ratio by volume of
70:29.5:0.5, respectively. A linear gradient (shown in the table
below) was used to separate procyanidin fractions using a 5 .mu.m
Supelcosil LC column (Silica, 100 Angstroms (.ANG.); 50.times.2 cm)
(Supelco, Inc., Bellefonte, Pa.) which was monitored by UV at a
wavelength of 280 nanometers (nm).
methylene chloride/ methanol/ time acetic acid/ water acetic acid/
water flow rate (minutes) (96:2:2 v/v)(%) (96:2:2 v/v)(%) (mL/min)
0 92.5 7.5 10 10 92.5 7.5 40 30 91.5 8.5 40 145 78.0 22.0 40 150
14.0 86.0 40 155 14.0 86.0 50 180 0 100 50
Fractions were collected at the valleys between the peaks
corresponding to oligomers. Fractions with equal retention times
from several preparative separations were combined, rotary
evaporated under partial vacuum and freeze dried.
Analysis of Purified Fractions by HPLC/MS
To determine purity of the individual oligomeric fractions, an
analysis was performed using a normal-phase high-performance
chromatograph (HPLC) method interfaced with online mass
spectrometry (MS) analysis using an atmospheric pressure ionization
electrospray (API-ES) chamber as described by Lazarus et al.
(1999), supra. Chromatographic analyses were performed on an HP
1100 series (Hewlett-Packard, Palo Alto, Calif.) equipped with an
auto-injector, quaternary HPLC pump, column heater, diode array
detector, and HP ChemStation for data collection and manipulation.
Normal-phase separations of the procyanidin oligomers were
performed on a Phenomenex (Torrance, Calif.) Luna silica column
(25.times.4.6 mm) at 37.degree. C. UV detection was recorded at a
wavelength of 280 nm. The ternary mobile phase consisted of (A)
dichloromethane, (B) methanol, and (C) acetic acid and water (1:1
v/v). Separations were effected by a series of linear gradients of
B into A with a constant 4% of (C) at a flow rate of 1 mL/min as
follows: elution starting with 14% of (B) into (A); 14-28.4% of (B)
into (A), 0-30 min; 28.4-50% of (B) into (A), 30-60 min; 50-86% of
(B) into (A), 60-65 min; and 65-70 min isocratic.
HPLC/MS analyses of purified fractions were performed using an HP
1100 series HPLC, as described above, and interfaced to an HP
series 1100 mass selective detector (model G1946A) equipped with an
API-ES ionization chamber. The buffering reagent was added via a
tee in the eluant stream of the HPLC just prior to the mass
spectrometer and delivered with an HP 1100 series HPLC pump,
bypassing the degasser. Conditions for analysis in the negative ion
mode included 0.75 M ammonium hydroxide as a buffering reagent at a
flow rate of 0.04 mL/min, a capillary voltage of 3 kV, a fragmentor
at 75 V, a nebulizing pressure of 25 psig, and a drying gas
temperature at 350.degree. C. Data were collected on an HP
ChemStation using both scan mode and selected ion monitoring (SIM).
Spectra were scanned over a mass range of m/z 100-3000 at 1.96
seconds per cycle. The ammonium hydroxide was used to adjust the
eluant pH to near neutrality via an additional auxiliary pump just
prior to entering the MS. This treatment counteracted the
suppression of negative ionization of the (-)-epicatechin standard
due to the elevated concentration of acid in the mobile phase. The
purity for each fraction was determined by peak area, using UV
detection at a wavelength of 280 nm in combination with a
comparison of the ion abundance ratio between each oligomeric
class.
Quantification of Procyanidins in Cocoa and Chocolate
A composite standard was made using commercially available
(-)-epicatechin for the monomer. Dimers through decamers were
obtained in a purified state by the methods described above.
Standard stock solutions using these compounds were analyzed using
the normal-phase HPLC method described above with fluorescence
detection at excitation and emission wavelengths of 276 nm and 316
nm, respectively. Peaks were grouped and their areas summed to
include contributions from all isomers within any one class of
oligomers and calibration curves generated using a quadratic fit.
Monomers and smaller oligomers had almost linear plots which is
consistent with prior usage of linear regression to generate
monomer-based and dimer-based calibration curves.
These calibration curves were then used to calculate procyanidin
levels in samples prepared as follows: First, the cocoa or
chocolate sample (about 8 grams) was de-fatted using three hexane
extractions (45 mL each). Next, one gram of de-fatted material was
extracted with 5 mL of the acetone/water/acetic acid mixture
(70:29.5:0.5 v/v). The quantity of procyanidins in the de-fatted
material was then determined by comparing the HPLC data from the
samples with the calibration curves obtained as described above
(which used the purified oligomers). The percentage of fat for the
samples (using a one gram sample size for chocolate or one-half
gram sample size for liquors) was determined using a standardized
method by the Association of Official Analytical Chemists (AOAC
Official Method 920.177). The quantity of total procyanidin levels
in the original sample (with fat) was then calculated. Calibration
was performed prior to each sample run to protect against
column-to-column variations.
EXAMPLE
Human volunteers were instructed to fast overnight and to maintain
low phytochemical intake the evening before the study.
Phytochemicals are components in plants and foods derived from
plants including many fruits, coffee, some teas, green peppers,
garlic, onions, yogurt, bran, and cruciferous vegetables such as
broccoli, cabbage, and cauliflower, etc.
Blood was drawn from the subjects prior to consumption of any food.
The subjects ingested either semisweet or dark chocolate. The two
different chocolates were used to demonstrate that the cocoa
polyphenols could be delivered in different forms and still exhibit
the same effects. The chocolates had different levels of chocolate
liquor and sugars as defined by the Standard of Identity rules for
semisweet chocolate and dark chocolate. The chocolate liquor used
to make these products was prepared from a blend of beans, some of
which were underfermented. After the initial blood was drawn, the
subjects were divided into two groups. One group was tested with
the semisweet chocolate and the other group was tested with the
dark chocolate. Both chocolates had enhanced levels of cocoa
procyanidins. The conserved levels were obtained by the process
described herein.
For the dark chocolate experiment, the control subjects consumed a
control bar which contained a low level of cocoa procyanidins,
i.e., only 3.3 mg cocoa procyanidins (1.8 mg monomer) per 36.9 gram
control product. The dark chocolate test product contained 147 mg
total cocoa procyanidins (40.6 mg monomer) per 36.9 gram test
product. Blood samples were drawn at 2 hours, after which another
bagel was consumed. At 6 hours, another blood sample was drawn.
FIG. 1 shows the nanomoles (nmol) of malondialdehyde (MDA) in
plasma at 2 and at 6 hours following ingestion of 1/2 bagel with
the dark chocolate test product or 1/2 bagel with the control
chocolate product having the low cocoa procyanidins. As
demonstrated by the data in FIG. 1, the higher the level of cocoa
procyanidins ingested, the lower the levels of MDA in the
plasma.
The control chocolate product which some of the subjects ingested
was prepared from jet black cocoa powder that was approximately ten
to twelve percent fat that was completely alkalized. The powder was
reconstituted in cocoa butter to give the proper percentage fat in
the dark chocolate test bar (taking into account the 9.87% fat in
the powder itself). The control bar was formulated with 49.335%
sugar, 19.75% jet black cocoa powder, 27.344% cocoa butter, 2.61%
anhydrous milk fat, 0.06% vanillin, 0.75% lecithin, 0.15% prova
vanilla, and 0.001% orange oil. The level of monomer was calculated
to be 1.8 mg per bar based upon the 3.3 mg per bar level of cocoa
procyanidins and the known levels of fat.
For the semisweet experiment, the control subjects consumed 1/2
bagel alone and no chocolate. The test group consumed 1/2 bagel
with one of three different chocolates, each with a different level
of cocoa procyanidins per bag. The first chocolate test product was
a 35 gram semisweet chocolate product containing 185 mg total cocoa
procyanidins (45.3 mg monomer) per 35 grams. The second chocolate
test product was a 70 gram semisweet chocolate product containing
370 mg total cocoa procyanidins. The third chocolate test product
was a 105 gram semisweet chocolate product containing 555 mg total
cocoa procyanidins. Blood samples were drawn at 2 hours, after
which another bagel was consumed. After 6 hours, another blood
sample was drawn. FIG. 2 shows the nanomoles (nmol) of
malondialdehyde (MDA) in plasma at 2 and at 6 hours following
ingestion of 1/2 bagel alone and following ingestion of 1/2 bagel
with increasing quantities of semisweet chocolate product, i.e.,
35, 70 and 105 grams, containing increasing quantities of total
cocoa procyanidins, i.e., 185, 370 and 555 mg. As demonstrated by
the data in FIG. 2, the higher the level of cocoa procyanidins
ingested, the lower the levels of MDA in the plasma.
For the analysis of the thiobarbituric reactive substances (TBARS),
a plasma sample (100 L) was mixed with 4% butylated hydroxytoluene
(BHT) and then frozen overnight. The sample was then thawed at room
temperature and a 100 L sample was mixed with 200 L sodium dodecyl
sulfate (SDS). The following reagents were then added in sequence:
800 L 0.1 N hydrochloric acid (HCl), 100 L 10%
1,4-benzenedicarboxylic acid (PTA), and 400 L 0.7% thiobarbituric
acid (TBA). The sample mixture was incubated in 95.degree. C. water
bath for 30 minutes. After cooling on ice, 1 ml of 1-butanol was
added. The sample was then centrifuged for 10 minutes at 1800 g
(3000 rpm) at 4 C. A 200 L aliquot of the butanol phase was assayed
for extracted MDA by fluorometry. This quantity was used for each
of the 96 wells of the plate which was read with excitation at 515
nm, slit 5 nm and emission at 555 nm, slit 5 nm.
The effect of the cocoa procyanidin levels on the oxidative stress,
as measured by the TBARS assay, was apparent at 2 hours and at 6
hours as shown by the change in total nanomoles of MDA per
milliliters of plasma. Whether the cocoa procyanidins were present
in the dark chocolate test product or in the semisweet chocolate
test products made no difference. In addition, the effect was more
pronounced as the amounts of total cocoa procyanidins consumed
increased.
* * * * *